Recombinant herpesvirus of turkeys (hvt) and preparation method and use thereof

ABSTRACT

The present disclosure provides a recombinant herpesvirus of turkeys (HVT) and a preparation method and use thereof. The present disclosure specifically provides a recombinant HVT, where an exogenous gene is inserted in a spacer region between an HVT005 region and an HVT006 region of an HVT genome; and the exogenous gene is selected from a gene derived from the group consisting of a Newcastle disease virus (NDV), an avian influenza virus (AIV), and an infectious bursal disease virus (IBDV); the spacer region between an HVT005 region and an HVT006 region of an HVT genome is located between 8,867 nt and 9,319 nt of the HVT genome, and has a nucleotide sequence set forth in SEQ ID NO: 1.

CROSS REFERENCE TO RELATED APPLICATION

This patent application claims the benefit and priority of ChinesePatent Application No. 202111318523.7, entitled “Recombinant Herpesvirusof Turkeys (HVT) and Preparation Method and Use Thereof” filed on Nov.9, 2021, the disclosure of which is incorporated by reference herein inits entirety as part of the present application.

TECHNICAL FIELD

The present disclosure belongs to the technical field of geneticengineering vaccines, in particular to a construction method of arecombinant herpesvirus of turkeys (HVT) strain using CRISPR-Cas9technology. The recombinant virus strain may provide desirableprotection as a vaccine.

BACKGROUND ART

As a recently emerging gene editing technology, the CRISPR-Cas9 systemhas achieved great success in efficient generation of geneticallyengineered cells and animal models. The CRISPR-Cas9 system is also usedto edit genomes of various large DNA viruses, including herpes simplexvirus, adenovirus, pseudorabies virus, poxvirus, guinea pigcytomegalovirus, and duck enteritis virus.

Herpesvirus of turkeys (HVT) has been widely used as a vaccine vector toexpress heterologous antigens of various avian diseases. These vaccinesprovide desirable and long-lasting immunity against the Marek's diseasevirus (MDV), and gene-dependent viruses inserted into vectors. However,these recombinant HVT vaccines are mainly produced by conventionalhomologous recombination in virus-infected cells, or by recombination inbacterial artificial chromosomes (BACs) from which a full-length genomeis cloned. These methods are inefficient for producing recombinantviruses, resulting in time-consuming production of the recombinantvaccines. It is of broad technical prospects in efficiently editinggenomes of avian herpesviruses using the CRISPR-Cas9 system to quicklygenerate recombinant viruses.

The insertion sites of HVT for exogenous genes that have been reportedinclude US2, US10, UL45/46, HVT065/066, and TK. The multiple defenseswith one vaccine can be achieved by discovering novel sites for HVT toexpress the exogenous genes, and constructing HVT-based live vectorvaccines inserting various exogenous genes based on the existinginsertion sites. This method has broad market prospects.

SUMMARY

An objective of the present disclosure is to provide a siteHVT005/HVT006 where an exogenous gene is inserted into a HVT.

An aspect of the present disclosure provides a recombinant HVT, in whichan exogenous gene is inserted in a spacer region between an HVT005region and an HVT006 region of an HVT genome; and the exogenous gene isselected from a gene derived from the group consisting of a Newcastledisease virus (NDV), an avian influenza virus (AIV), and an infectiousbursal disease virus (IBDV);

in some embodiments, the AIV is selected from an H9N2 subtype AIV, anH5N1 subtype AIV, an H7N7 subtype AIV, an H5N2 subtype AIV, an H7N2subtype AIV, and an H9N1 subtype AIV; and the NDV is selected from a VIItype NDV, a II type NDV, and a III type NDV; and

in some embodiments, the exogenous gene is selected from an NDV F gene,an AIV HA gene, and an IBDV VP2 gene.

In some embodiments, the spacer region between an HVT005 region and anHVT006 region of an HVT genome is located between 8,867 nt and 9,319 ntof the HVT, and has a nucleotide sequence preferably set forth in SEQ IDNO: 1.

In some embodiments, the NDV F gene is a complete open reading frame(ORF) of a VII type NDV F gene, and has a cleavage site replaced with acleavage site of a live vaccine (La Sota strain) of Newcastle disease inchicken.

In some embodiments, the nucleotide sequence of the NDV F gene has anucleotide sequence set forth in SEQ ID NO: 2.

In some embodiments, an expression cassette of the NDV F gene is ligatedsuccessively by an mCMV promoter, the NDV F gene and an SV40 poly A.

In some embodiments, the AIV HA gene is a complete ORF of an H9N2subtype AIV HA gene.

In some embodiments, the nucleotide sequence of the H9N2 subtype AIV HAgene is set forth in SEQ ID NO: 3.

In some embodiments, an expression cassette of the H9N2 subtype AIV HAgene is ligated successively by an mCMV promoter, the exogenous gene andan SV40 poly A.

The present disclosure further provides a method for preparing therecombinant HVT, including the following steps:

S1) Construction of a CRISPR-Cas9 plasmid:

S1-1) designing an sgRNA sequence for the spacer region between anHVT005 region and an HVT006 region, in which the sgRNA sequence is setforth in SEQ ID NO: 7, TCATATACTGAATCGTAGGG (SEQ ID NO: 7); and

S1-2) synthesizing a plus-strand HVT005/006-sgRNA-F and a minus-strandHVT005/006-sgRNA-R according to the designed sgRNA sequence, andconducting annealing to form a dsDNA, and inserting the dsDNA into avector to obtain the CRISPR-Cas9 plasmid; in which

sequences of the plus-strand HVT005/006-sgRNA-F and the minus-strandHVT005/006-sgRNA-R are set forth in SEQ ID NO: 8 and SEQ ID NO: 9,respectively:

Name Sequence HVT005/006-sgRNA-F caccgCATATACTGAATCGTAGGG SEQ ID NO: 8HVT005/006-sgRNA-R aaacCCCTACGATTCAGTATATGc SEQ ID NO: 9

S2) constructing a donor vector plasmid including an expression cassetteof the exogenous gene;

S3) construction and purification of a recombinant HVT: co-transfectinga pX459-sgA plasmid, the CRISPR-Cas9 plasmid and the donor vectorplasmid into primary chicken embryo fibroblasts (CEFs), inoculating withan HVT, followed by culturing until the primary CEFs are cytopathic; andconducting purification to obtain a primary recombinant HVT; and

S4) removing a green fluorescent protein (GFP) in the primaryrecombinant HVT obtained in step S3) to obtain the recombinant HVT.

In some embodiments, step S2) includes specifically the following steps:

S2-1) inserting the exogenous gene into a pcDNA3.1-SfiI vector to obtaina pcDNA3.1-SfiI-exogenous gene vector; and

S2-2) ligating an expression cassette of the exogenous gene in thepcDNA3.1-SfiI-exogenous gene vector into a pT-sgA-GFP vector through anSfiI restriction site to obtain a pT-sgA-GFP-exogenous gene vector.

In some embodiments, step S4) includes specifically the following steps:transfecting a pcDNA3.1-Cre plasmid in the primary CEFs, inoculatingwith the primary recombinant HVT obtained in step S3) and conductingculture until the primary CEFs are cytopathic, followed by purificationthrough virus spotting to obtain a recombinant virus rHVT-exogenousgene.

The present disclosure further provides use of the recombinant HVT inpreparation of a vaccine for preventing avian influenza and/or Newcastledisease and/or infectious bursal disease.

The present disclosure further provides a vaccine for preventing avianinfluenza and/or Newcastle disease and/or infectious bursal disease,including the recombinant HVT.

The present disclosure further provides a method for inserting anexogenous gene in an HVT, including inserting the exogenous gene into anHVT genome using a CRISPR-Cas9 technology; in which

the exogenous gene is inserted into a spacer region between an HVT005region and an HVT006 region of the HVT genome;

In some embodiments, an insertion site is located between 8,867 nt and9,319 nt in the spacer region between an HVT005 region and an HVT006region of the HVT genome, and the nucleotide sequence of the insertionis set forth in SEQ ID NO: 1; and

In some embodiments, the CRISPR-Cas9 technology is achieved byconstruction of a CRISPR-Cas9 plasmid, including:

S1-1) designing an sgRNA sequence for the spacer region between anHVT005 region and an HVT006 region, in which the sgRNA sequence is setforth in SEQ ID NO: 7, TCATATACTGAATCGTAGGG SEQ ID NO: 7; and

S1-2) synthesizing a plus-strand HVT005/006-sgRNA-F and a minus-strandHVT005/006-sgRNA-R according to the designed sgRNA sequence, andconducting annealing to form a dsDNA, and inserting the dsDNA into avector to obtain the CRISPR-Cas9 plasmid; where

sequences of the plus-strand HVT005/006-sgRNA-F and the minus-strandHVT005/006-sgRNA-R are set forth in SEQ ID NO: 8 and SEQ ID NO: 9,respectively:

Name Sequence HVT005/006-sgRNA-F caccgCATATACTGAATCGTAGGG SEQ ID NO: 8HVT005/006-sgRNA-R aaacCCCTACGATTCAGTATATGc SEQ ID NO: 9

The present disclosure provides a site for inserting an exogenous geneinto an HVT genome, that is, the exogenous gene is inserted into aspacer region between an HVT005 region and an HVT006 region of the HVTgenome. More specifically, the insertion site is located between 8,867nt and 9,319 nt in the spacer region between an HVT005 region and anHVT006 region of the HVT genome, and has a nucleotide sequence set forthin SEQ ID NO: 1.

The present disclosure provides a CRISPR/Cas9-based sgRNA guide sequenceHVT005/006-sgRNA targeting a HVT005-HVT006 spacer region.

HVT005/006-sgRNA: (SEQ ID NO: 7) TCATATACTGAATCGTAGGG

In the present disclosure, the NDV F gene is a complete ORF of a VIItype NDV F gene, and has a cleavage site replaced with a cleavage siteof a live vaccine (La Sota strain) of Newcastle disease in chicken. Thenucleotide sequence of the NDV F gene is set forth in SEQ ID NO: 2. Anexpression cassette of the NDV F gene is ligated successively by an mCMVpromoter, the exogenous gene and an SV40 poly A.

In the present disclosure, the AIV HA gene is a complete ORF of an H9N2subtype AIV HA gene, having a nucleotide sequence set forth in SEQ IDNO: 3. In some embodiments, an expression cassette the H9N2 subtype AIVHA gene is ligated successively by an mCMV promoter, the exogenous geneand an SV40 poly A.

The mCMV promoter is a mouse-derived mCMV promoter, with a nucleotidesequence set forth in SEQ ID NO: 4. The nucleotide sequence of the SV40poly A is set forth in SEQ ID NO: 5.

A construction method of a recombinant HVT expressing an NDV F protein,including the following steps: inserting an exogenous gene into an HVTgenome using CRISPR-Cas9 technology, and conducting screening andpurification to obtain a recombinant virus, namely the recombinant HVTexpressing an NDV F protein.

The construction method of the recombinant HVT expressing an NDV Fprotein includes specifically the following steps:

(1) construction of a CRISPR-Cas9 plasmid, pX330-HVT05/06-sgRNA:

1) designing and synthesizing an sgRNA sequence for an HVT005/006 targetsite; in which a specific sequence is HVT005/006-sgRNA:TCATATACTGAATCGTAGGG (SEQ ID NO: 7);

2) annealing synthesized HVT005/006-sgRNA-F and HVT005/006-sgRNA-Rsequences to form a dsDNA, and inserting the dsDNA into a pX330 vector(purchased from YouBio) to construct the pX330-HVT05/06-sgRNA;

(2) construction of a donor vector pT-sgA-GFP-F:

1) inserting an F gene fragment into a pcDNA3.1-SfiI vector (referringto 2.2.2.1 of Example) to obtain a pcDNA3.1-SfiI-F vector; and

2) ligating an expression cassette of the F gene in the pcDNA3.1-SfiI-Fvector into a pT-sgA-GFP vector through an SfiI restriction site(referring to 2.2.3 of Example) to obtain the pT-sgA-GFP-F vector; and

(3) construction and purification of a recombinant HVT, rHVT-F:

1) co-transfecting a pX459-sgA plasmid (referring to 1.1 of Example), apT-sgA-GFP-F plasmid (referring to 2.2 of Example) and apX330-HVT005/006-sgRNA plasmid (referring to 2.1 of Example) intoprimary chickens embryo fibroblasts (CEFs), inoculating with an HVTFC126 strain, conducting culture at 37° C. until the primary CEFs arecytopathic, followed by purification through virus spotting to obtain arecombinant virus rHVT-GFP-F; and

2) transfecting a plasmid pcDNA3.1-Cre (purchased from YouBio) into theprimary CEFs, inoculating with the recombinant virus rHVT-GFP-F,conducting culture at 37° C. until the primary CEFs are cytopathic,followed by conducting purification through virus spotting to obtain therecombinant virus rHVT-F, namely the recombinant HVT expressing an NDV Fprotein.

A construction method of a recombinant HVT expressing an AIV HA proteinis the same as the construction method of a recombinant HVT expressingan NDV F protein but the F gene is replaced by the HA gene. Otherpathogen-related protective antigen genes can also be constructedaccording to the same method.

Beneficial Effects

1. In the present disclosure, it is found for the first time that thespacer region between the HVT005 region and the HVT006 region can beused as an insertion site for the exogenous gene of the HVT. The NDV Fgene and the AIV HA gene are inserted into the spacer region between theHVT005 region and the HVT006 region of the HVT genome using CRISPR/Cas9technology, and a vaccine candidate is obtained for prevention ofNewcastle disease and avian influenza in chicken.

2. Compared with common homologous recombination, BAC homologousrecombination and other technical means, the CRISPR/Cas9 gene knock-intechnology is more effective and rapid, which is helpful for rapidconstruction of an MDV-based recombinant vaccine.

3. The CRISPR/Cas9 is a simple knock-in method, and the designed sgRNAsequence along with the donor plasmid may efficiently insert the targetsites.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 a -If show a schematic diagram of construction of a donorplasmid;

FIG. 2 shows a schematic diagram of construction of a recombinant virusrHVT-F;

FIG. 3 shows a schematic diagram of construction of a recombinant virusrHVT-HA;

FIG. 4 shows a PCR electrophoresis identification map of apX330-HVT005/006-sgRNA plasmid;

FIG. 5 shows a fluorescence image of a recombinant virus rHVT-GFP-F;

FIG. 6 shows a fluorescence image of a recombinant virus rHVT-GFP-HA;

FIG. 7 shows a fluorescence image of the recombinant virus rHVT-Fidentified by indirect immunofluorescence (IFA);

FIG. 8 shows a fluorescence image of the recombinant virus rHVT-HAidentified by IFA;

FIG. 9 shows a PCR electrophoresis image of PCR identification ofpassage stability of the recombinant virus rHVT-F; and

FIG. 10 shows a PCR electrophoresis image of PCR identification ofpassage stability of the recombinant virus rHVT-HA.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present disclosure will be described in detail below by takingconstruction of rHVT-F and rHVT-HA as an example.

EXAMPLE

1. Materials

1.1. Strains, Plasmids and Antibodies

An HVT FC-126 vaccine strain, an NDV genotype VII JS strain and an AIVH9N2 (2019) strain were identified and preserved by the Key Laboratoryof Avian Preventive Medicine, Ministry of Education, YangzhouUniversity. All the strains were purchased therefrom.

Plasmids: pX330, pX459, pcDNA3.1 and pcDNA3.1-Cre plasmids werepurchased from a website of Hunan Keai Medical Equipment Co., Ltd.(YouBio). The pGEM®-T Easy Vector Systems were purchased from Promega. Aplasmid pCMV-N-GFP was purchased from Beyotime Biotechnology. A plasmidpX459-sgA was constructed and preserved by the Key Laboratory of AvianPreventive Medicine, Ministry of Education, Yangzhou University; theplasmid pX459-sgA was obtained by ligating an sgA sequence into thepX459 plasmid through a BbsI restriction site (referring to Knock-in oflarge reporter genes in human cells via CRISPR/Cas9-inducedhomology-dependent and independent DNA repair,https://doi.org/10.1093/nar/gkw064).

Antibodies: anti-HA (H9) monoclonal antibody 2G4 (referring to“Identification and Analysis of Critical Amino Acids Mutations inEpitopes in Hemagglutinin and Neuraminidase of H9N2 Influenza Virus inthe Pressure of Antibody”, Zhimin Wan) and anti-F (NDV) monoclonalantibody 1D10 (referring to “Characteristics and Preliminary Applicationof Anti-Newcastle Disease Virus Fusion Protein Monoclonal Antibody”,Qiangian Wang) were prepared and preserved by the Key Laboratory ofAvian Preventive Medicine, Ministry of Education, Yangzhou University.

2. Methods

2.1. Target Site Design and sgRNA Sequence Synthesis

An exogenous gene was inserted into an HVT005-HVT006 spacer regionwithin the HVT; referring to an HVT005-HVT006 spacer region sequence ofa full gene sequence of an HVT FC126 strain (accession number: AF291866)registered in the GenBank, a target-site-based sgRNA was designed usinga sgRNA design website (http://crispr.mit.edu/) to obtain a sgRNAsequence.

Name Sequence HVT005/006-SgRNA TCATATACTGAATCGTAGGG SEQ ID NO: 7

A base CACC was added at 5′-end of the sgRNA guide sequence, and a firstbase T was replaced with G to form a plus-strand sgRNA sequence; thedesigned sgRNA sequence was reverse-complemented, and a base AAAC wasadded to the 5′-end to form a minus-strand sgRNA sequence.

Name Sequence HVT005/006-sgRNA-F caccgCATATACTGAATCGTAGGG SEQ ID NO: 8HVT005/006-sgRNA-R aaacCCCTACGATTCAGTATATGc SEQ ID NO: 9

2.2. Construction of a Targeting Vector:

(1) synthesized HVT005/006-sgRNA-F and HVT005/006-sgRNA-R sequences wereannealed to form a dsDNA, in which a reaction system was as follows:

Name Volume HVT005/006-sgRNA-F(10 μM) 1 μL HVT005/006-sgRNA-R(10 μM) 1μL Annealing buffer (10×) 1 μL ddH₂O 7 μL

The above system was shaken, centrifuged and placed in a PCR instrument.The PCR protocol consisted of 30 min at 37° C., 5 min at 95° C.,gradient cooling at 5° C./min, and then 10 min at 4° C.

(2) The pX330 vector was digested with a BbsI enzyme for linearization,in which a reaction system was as follows:

Name Volume Vector pX330 1 μg 10 × NEB Buffer 2 μL BbsI enzyme 1 μLddH₂O Adding to 20 μL

After incubation at 37° C. for 2 h, a linearized vector pX330 wasexcised and recovered.

(3) An annealed dsDNA was ligated with the linearized vector pX330, inwhich a reaction system was as follows:

Name Volume Linearized vector pX330 0.1 μg dsDNA 0.2 μL 10 × ligationbuffer 2 μL T4 DNA ligase 1 μL ddH₂O Adding to 20 μL

The above system was shaken, centrifuged, and ligated at 16° C.overnight to obtain a ligated product.

(4) 50 μL of DH5α competent cells (purchased from Nanjing Vazyme BiotechCo., Ltd.) were mixed well with 10 μL of the ligated product, followedby conducting incubation on ice for 30 min. The above mixture wastreated in a water bath at 42° C. for 90 sec, and a tube was quicklyinserted into ice and allowed to stand for 3 min. 800 μL of ananti-LB-free medium was added to the tube, followed by incubation on ashaker (37° C./150 rpm) for 1 h; 200 μL of a bacterial solution waspipetted and spread evenly on a solid medium containing ampicillin (50μg/mL) prepared in advance, followed by inverted incubation at 37° C.,and transformed clones were formed at 14 h to 16 h.

(5) A plasmid was extracted, and primers were designed according to thevector pX330 and the sgRNA sequence for PCR identification.

Name Sequence U6-F GACTATCATATGCTTACCGT SEQ ID NO: 10 HVT005/006-SgRNA-RaaacCCCTACGATTCAGTATATGc SEQ ID NO: 11

PCR System

Name Volume U6-F 1 μL HVT005/006-sgRNA-R 1 μL Plasmid 1 μL ddH₂O 7 μLGreen Taq Mix (Vazyme Biotech) 10 μL 

The reaction program was as follows: 95° C. for 5 min; 35 cycles of 95°C. for 30 sec, 60° C. for 30 sec, and then 72° C. for 30 sec; and 72° C.for 10 min.

After the reaction, electrophoresis was conducted with 1% agarose gel(FIG. 3 ).

2.2. Construction of a Donor Vector pT-sgA-GFP-F

2.2.1. Amplification of NDV F Gene

Overlap PCR primers were designed for an F gene sequence of an NDVgenotype VII and an F gene cleavage site sequence of an NDV La Sotastrain. Primer sequences were as follows:

Name Sequence F(overlap)left-F ATGGGCTCCAAACTTTCTAC SEQ ID NO: 12F(overlap)left-R GCGCCCCTGTCTCCC TCCTCCAGACGTGGACAC SEQ ID NO: 13F(overlap)right-F GAGGGAGACAGGGGCGCCTTATAGGTGCTGTTATTGGCAG SEQ ID NO: 14F(overlap)right-R TCATGCTCTTGTAGTGGCTC SEQ ID NO: 15

PCR amplification was conducted on a left fragment of the F gene and aright fragment of the F gene taking a reverse-transcribed cDNA of theNDV genotype VII as a template, and using F(overlap)left-F andF(overlap)left-R, F(overlap)right-F and F(overlap)right-R as primers,respectively; and overlap PCR amplification was conducted on an F genefragment after replacement of the cleavage site using the left fragmentof the F gene and the right fragment of the F gene as templates, andusing F(overlap)left-F and F(overlap)right-R were used as primers. The Fgene fragment after replacement of the cleavage site had a nucleotidesequence set forth in SEQ ID NO: 2.

Primers were designed with Primer Premier 5.0 software, and an NotIrestriction sites were introduced upstream and downstream. Primersequences were as follows:

Name Sequence NotI-F-F ATTTGCGGCCGCATGGGCTCCAAACTTTCTACCA SEQ ID NO: 16NotI-F-R ATTTGCGGCCGCTCATGCTCTTGTAGTGGCTCTCA SEQ ID NO: 17

The PCR amplification was conducted on the F gene using the F genefragment after replacement of the cleavage site as a template, and usingNotI-F-F and NotI-F-R as primers; electrophoresis was conducted with 1%agarose gel after the reaction, and a target band was recovered forlater use.

2.2.2. Construction of an Eukaryotic Expression Vector pcDNA3.1-SfiI-F

2.2.2.1. Construction of a Plasmid pcDNA3.1-SfiI

An mCMV+polyA element for gene sequence synthesis(NheI+SfiI+mCMV+NotI+SV40ployA+SfiI+PmeI) was synthesized, in which anmCMV sequence referred to a mouse cytomegalovirus genome (Muridherpesvirus 1 strain Smith, complete genome, GenBank: GU305914.1,184,336 nt to 182,946 nt), and the entire element had a nucleotidesequence set forth in SEQ ID NO: 6.

The sequence fragment of mCMV+polyA for gene synthesis wasdouble-digested with NheI and PmeI, and ligated into a pcDNA3.1 plasmidthrough NheI and PmeI restriction sites to obtain a plasmidpcDNA3.1-SfiI.

2.2.2.2. Construction of a Plasmid pcDNA3.1-SfiI-F

PCR gel recovery products of the F gene in 2.2.1 and the plasmidpcDNA3.1-SfiI were digested with the NotI; after conducting 1% gelelectrophoresis, the gel was cut to recover a linearizedpcDNA3.1-SfiI-NotI vector and an NotI-F gene fragment. The recoveredpcDNA3.1-SfiI-NotI fragment and the NotI-F gene fragment were ligated toconstruct the plasmid pcDNA3.1-SfiI-F.

2.2.3. Construction of a Donor Vector pT-sgA-GFP-F

2.2.3.1. Construction of a Plasmid pT-sgA

A gene sequence (sgA+loxP+PacI+LoxP+SfiI+spacer+SfiI+sgA) wassynthesized, in which primers were as follows; sgA-SfiI-F and sgA-SfiI-Rwere annealed to form dsDNA fragments, and the fragments were ligatedinto a pGEM-T-easy vector to obtain a pT-sgA vector.

Name Sequence sgA- GAGATCGAGTGCCGCATCACCGGATAACTTCGTATAATGTATGCTATACGAASfiI-F GTTATTTAATTAAATAACTTCGTATAATGTATGCTATACGAAGTTATGGCCGCCTAGGCCGGCGCGCCGTTTAAACGGCCATTATGGCCGAGATCGAGTGCCGCATCACCGGA SEQ ID NO: 18 sgA-CCGGTGATGCGGCACTCGATCTCGGCCATAATGGCCGTTTAAACGGCGCGC SfiI-RCGGCCTAGGCGGCCATAACTTCGTATAGCATACATTATACGAAGTTATTTAATTAAATAACTTCGTATAGCATACATTATACGAAGTTATCCGGTGATGCGGCACTCGATCTCA SEQ ID NO: 19

2.2.3.2. Construction of a Plasmid pT-sgA-GFP

The following primers were designed for a GFP expression cassette:

Name Sequence PacI-GFP-FCCTTAATTAAGGTTAATTAA TTTGCTGGCCTTTTGCTCAC SEQ ID NO: 20 PacI-GFP-RCCTTAATTAAGGTTAATTAA GCCGATTTCGGCCTATTGGT SEQ ID NO: 21

The GFP expression cassette (PacI+CMV promoter+GFP+SV40PolyA+PacI) wasamplified using a pCMV-N-GFP plasmid as a template, and using PacI-GFP-Fand PacI-GFP-R as primers, and then ligated through a PacI restrictionsite into a pT-sgA plasmid to obtain the pT-sgA-GFP plasmid.

2.2.3.3. Construction of a Plasmid pT-sgA-GFP-F

The plasmids pcDNA3.1-SfiI-F and pT-sgA-GFP were digested with the SfiI,followed by conducting 1% gel electrophoresis, and an SfiI-F fragment inthe pcDNA3.1-SfiI-F vector and a linearized pT-sgA-GFP vector wererecovered by gel cutting. The recovered SfiI-F fragment and thepT-sgA-GFP-SfiI fragment were ligated to construct the plasmidpT-sgA-GFP-F.

2.3. Construction of a Donor Vector pT-sgA-GFP-HA

2.3.1. Amplification of AIV HA Gene

Primers were designed with Primer Premier 5.0 software, and an NotIrestriction sites were introduced upstream and downstream. Primersequences were as follows:

Name Sequence NotI-HA-FATTTGCGGCCGCATGGAGGCAGTATCACTAATAAC SEQ ID NO: 22 NotI-HA-RATTTGCGGCCGC TTATATACAAATGTTGCATCTGC SEQ ID NO: 23

The PCR amplification was conducted on the HA gene using an AIV H9N2(2019) strain cDNA as a template, and using NotI-HA-F and NotI-HA-R asprimers; 1% agarose gel electrophoresis was conducted after thereaction, and a target band was recovered for later use.

The HA gene had a nucleotide sequence set forth in SEQ ID NO: 3.

2.3.2. Construction of an Eukaryotic Expression Vector pcDNA3.1-SfiI-HA

PCR gel recovery products of the HA gene in 2.3.1 and the plasmidpcDNA3.1-SfiI (referring to 2.2.2.1 in Example) were digested with theNotI; after conducting 1% gel electrophoresis, the gel was cut torecover a linearized pcDNA3.1-SfiI vector and an NotI-HA gene fragment.The recovered pcDNA3.1-SfiI-NotI fragment and the NotI-HA fragment wereligated to construct the plasmid pcDNA3.1-SfiI-HA.

2.3.3. Construction of a Donor Vector pT-sgA-GFP-HA

The plasmids pcDNA3.1-SfiI-HA and pT-sgA-GFP (referring to 2.2.3.2 inExample) were digested with the SfiI, followed by conducting 1% gelelectrophoresis, and an SfiI-HA fragment in the pcDNA3.1-SfiI-HA vectorand a linearized pT-sgA-GFP vector were recovered by gel cutting. Therecovered SfiI-HA fragment and the pT-sgA-GFP-SfiI fragment were ligatedto construct the plasmid pT-sgA-GFP-HA.

2.4. Construction of a Recombinant HVT for Expressing GFP and FProteins, rHVT-GFP-F

2.4.1. Cell Transfection

Before transfection, primary CEF cells were prepared, in which the cellswere grown in a 24-well cell culture plate with an M199 mediumcontaining 5% fetal bovine serum; when the cells grew to 80% of amonolayer, they were transfected with TransIT-X2 according toinstructions of the TransIT-X2 transfection reagent (Mirusbio), in whicha transfection system of each well included 0.25 μg of plasmidpX459-sgA, 0.25 μg of plasmid pT-sgA-GFP-F and 0.25 μg of plasmidpX330-HVT005/006-sgRNA.

2.4.2. Virus Infection

8 h to 12 h after the transfection, the cells were infected with theHVT, in which the virus in each well had an amount of 5,000 PFU. Afterincubation for 2 d to 3 d at 37° C. and 5% CO₂, cells in 1 well weredigested with trypsin and inoculated on 2 new CEF 6-well cell cultureplates. After incubation for 3 d to 4 d at 37° C. and 5% CO₂, the cellswere observed under a fluorescence microscope, and viral plaques wereselected with green fluorescence.

2.4.3. Purification of a Recombinant Virus rHVT-GFP-F

The cells were observed under the fluorescence microscope, the viralplaques with green fluorescence were marked using a marker, and labeledgreen fluorescence-containing cells were selected by trypsin digestion,and inoculated on a secondary CEF monolayer cells, followed byincubation at 37° C. and 5% CO₂ for 3 d to 4 d; the above steps wererepeated until all plaques showed fluorescence, and acompletely-purified recombinant virus was named rHVT-GFP-F (shown inFIG. 5 ).

2.5. Construction of a Recombinant HVT for Expressing GFP and HAProteins, rHVT-GFP-HA

2.5.1. Cell Transfection

Before transfection, primary CEF cells were prepared, in which the cellswere grown in a 24-well cell culture plate with an M199 mediumcontaining 5% fetal bovine serum; when the cells grew to 80% of amonolayer, they were transfected with TransIT-X2 according toinstructions of the TransIT-X2 transfection reagent (Mirusbio), in whicha transfection system of each well included 0.25 μg of plasmidpX459-sgA, 0.25 μg of plasmid pT-sgA-GFP-HA and 0.25 μg of plasmidpX330-HVT005/006-sgRNA.

2.5.2. Virus Infection

8 h to 12 h after the transfection, the cells were infected with theHVT, in which the virus in each well had an amount of 5,000 PFU. Afterincubation for 2 d to 3 d at 37° C. and 5% CO₂, cells in 1 well weredigested with trypsin and inoculated on 2 new CEF 6-well cell cultureplates. After incubation for 3 d to 4 d at 37° C. and 5% CO₂, the cellswere observed under a fluorescence microscope, and viral plaques wereselected with green fluorescence.

2.5.3. Purification of a Recombinant Virus rHVT-GFP-HA

The cells were observed under the fluorescence microscope, the viralplaques with green fluorescence were marked using a marker, and labeledgreen fluorescence-containing cells were selected by trypsin digestion,and inoculated on a secondary CEF monolayer cells, followed byincubation at 37° C. and 5% CO₂ for 3 d to 4 d; the above steps wererepeated until all plaques showed fluorescence, and acompletely-purified recombinant virus was named rHVT-GFP-HA (shown inFIG. 6 ).

2.6. Construction of a Recombinant HVT for Expressing an F Protein,rHVT-F

2.6.1. Cell Transfection

Before transfection, primary CEF cells were prepared, where the cellswere grown in a 24-well cell culture plate with an M199 mediumcontaining 5% fetal bovine serum; when the cells grew to 80% of amonolayer, they were transfected with TransIT-X2 according toinstructions of the TransIT-X2 transfection reagent (Mirusbio), in whicha transfection system of each well included 0.5 μg of plasmidpcDNA3.1-Cre.

2.6.2. Virus Infection

8 h to 12 h after the transfection, the cells were infected with therHVT-GFP-F, in which the virus in each well had an amount of 5,000 PFU.After incubation for 2 d to 3 d at 37° C. and 5% CO₂, cells in 1 wellwere digested with trypsin and inoculated on 2 new CEF 6-well cellculture plates. After incubation for 3 d to 4 d at 37° C. and 5% CO₂,the cells were observed under a fluorescence microscope, and viralplaques were selected without green fluorescence.

2.6.3. Purification of a Recombinant Virus rHVT-F

The cells were observed under the fluorescence microscope, the viralplaques without green fluorescence were marked using a marker, andlabeled pathological cells were selected by trypsin digestion, andinoculated on a secondary CEF monolayer cells, followed by incubation at37° C. and 5% CO₂ for 3 d to 4 d; the above steps were repeated untilall plaques showed no fluorescence, and a completely-purifiedrecombinant virus was named rHVT-F.

2.7. Construction of a Recombinant HVT for Expressing an HA Protein,rHVT-HA

2.7.1. Cell Transfection

Before transfection, primary CEF cells were prepared, in which the cellswere grown in a 24-well cell culture plate with an M199 mediumcontaining 5% fetal bovine serum; when the cells grew to 80% of amonolayer, they were transfected with TransIT-X2 according toinstructions of the TransIT-X2 transfection reagent (Mirusbio), in whicha transfection system of each well included 0.5 μg of plasmidpcDNA3.1-Cre.

2.7.2. Virus Infection

8 h to 12 h after the transfection, the cells were infected with therHVT-GFP-HA, in which the virus in each well had an amount of 5,000 PFU.After incubation for 2 d to 3 d at 37° C. and 5% CO₂, cells in 1 wellwere digested with trypsini and inoculated on 2 new CEF 6-well cellculture plates. After incubation for 3 d to 4 d at 37° C. and 5% CO₂,the cells were observed under a fluorescence microscope, and viralplaques were selected without green fluorescence.

2.7.3. Purification of a Recombinant Virus rHVT-HA

The cells were observed under the fluorescence microscope, the viralplaques without green fluorescence were marked using a marker, andlabeled pathological cells were selected by trypsin digestion, andinoculated on a secondary CEF monolayer cells, followed by incubation at37° C. and 5% CO₂ for 3 d to 4 d; the above steps were repeated untilall plaques showed no fluorescence, and a completely-purifiedrecombinant virus was named rHVT-HA.

2.8. Identification of the Recombinant Virus and Detection of PassageStability

2.8.1. Indirect Immunofluorescence Assay (IFA)

The recombinant virus rHVT-F strain, the recombinant virus rHVT-HAstrain and an HVT parental virus were inoculated into a 24-well cellculture plate covered with monolayer CEF, followed by incubation at 37°C. and 5% CO₂; after the cells had typical plaques, the medium wasdiscarded, and the cells were fixated with a cold fixative solution ofacetone:ethanol (3:2) for 10 min, and washed twice with a PBS; 500 μL(1:200 dilution) of a 1D10 monoclonal antibody was added to the wellsinoculated with the rHVT-F strain, 500 μL (1:200 dilution) of a 2G4monoclonal antibody was added to the wells inoculated with the rHVT-HAstrain, 500 μL (1:200 dilution) of the 1D10 monoclonal antibody wasadded to one well inoculated with the HVT strain, and 500 μL (1:200dilution) of the 2G4 monoclonal antibody was added to the other wellinoculated with the HVT strain. The cells were incubated for 1 h in aconstant-temperature incubator at 37° C., and washed three times withthe PBS; 500 μL (1:800 dilution) of an Alexa Fluor® 594 goat anti-mouseIgG antibody was added to each well, followed by incubation in a 37° C.incubator for 1 h, and then washing three times with the PBS; the cellswere observed under a fluorescence microscope. It was showed thatspecific red fluorescence appeared in the wells infected with the rHVT-Fand rHVT-HA strains (shown in FIG. 7 and FIG. 8 ), while HVT-infectedwells had no fluorescence, indicating that the F gene of rHVT-F strainand the HA gene of rHVT-HA strain were correctly expressed.

2.8.2. PCR Detection of the Passage Stability

The rHVT-F strain and the rHVT-HA strain were continuously passed on CEFfor 15 generations, and the genome of the recombinant virus wasextracted every 5 generations; PCR was conducted using the extractedgenome as a template and HVT-005/006-F and HVT-005/006-R as primers, andtarget bands of about 3,500 bp were amplified, respectively (shown inFIG. 9 and FIG. 10 ). The results showed that the F gene and the HA genewere successfully inserted into the HVT genome without gene loss.

Name Sequence HVT-005/006-F tcgtttgcgcgtagtaacatt SEQ ID NO: 24HVT-005/006-R taactgtgagcaatgcagggg SEQ ID NO: 25

3. The rHVT-F Strain as a Vaccine

3.1. Amplification of the rHVT-F Strain

CEFs were inoculated into several T75 cell flasks, and inoculated withrHVT-F after 1 d of incubation, and each flask was inoculated with50,000 PFU of the virus. After inoculation, the virus growth wasobserved every day; generally, 48 h to 72 h after inoculation, when morethan 70% of the monolayer cells had typical cytopathic effects, the cellmedium was discarded, and after washing with PBS once, the cells weredigested with an appropriate amount of trypsin; when the monolayer cellsshrank and were about to separate from the bottle wall, the trypsin wasdiscarded, and the digestion was terminated using an equal amount of acell medium containing 5% bovine serum. The cells were pipetted with apipette to make the cells all detached from the bottle wall; after beingcollected by centrifugation, the cells were resuspended with acryoprotectant (a cell medium containing 15% bovine serum and 10% DMSO),divided into cryovials at −70° C., and transferred to liquid nitrogenfor storage on a second day.

3.2. Immunization of the rHVT-F Strain

18 1-day-old SPF chickens were randomly divided into two groups, namelyan rHVT-F group and a challenge control group; in which the rHVT-F groupwas inoculated with 2,000 PFU/chicken of an rHVT-F recombinant vaccineby intraperitoneal injection at 1 day old, while the challenge controlgroup was inoculated with a vaccine dilution. The two groups of chickenswere challenged with a virulent NDV F48E8 strain by intramuscularinjection at 28-day-old, with a challenge dosage of 10⁵ ELD₅₀/chicken.After the challenge, the incidence and mortality of the chickens in eachgroup were observed and counted every day for two weeks.

3.3. Challenge Protection Effects of rHVT-F Strain-Immunized Chickens

The incidence and mortality of the chickens in each group are shown inthe table below. It could be seen that the chickens in the rHVT-F grouphad a challenge protection rate reaching 100%, while the challengecontrol group had a challenge protection rate of 0%.

Challenge protection Group Mortality (%) efficiency rHVT-F 0/9 100%Challenge control group 9/9  0%

The results of immune protection evaluation showed that the chickens hada desirable immune protection effect against the virulent NDV strainafter immunization with the recombinant HVT, rHVT-F strain.

What is claimed is:
 1. A recombinant herpesvirus of turkeys (HVT),wherein an exogenous gene is inserted in a spacer region between anHVT005 region and an HVT006 region of an HVT genome; and the exogenousgene is selected from a gene derived from the group consisting of aNewcastle disease virus (NDV), an avian influenza virus (AIV), aninfectious bursal disease virus (IBDV), and a protective gene of otherepidemic disease pathogens of chicken; the AIV is selected from an H9N2subtype AIV, an H5N1 subtype AIV, an H7N7 subtype AIV, an H5N2 subtypeAIV, an H7N2 subtype AIV, and an H9N1 subtype AIV; and the NDV isselected from a VII type NDV, a II type NDV, and a III type NDV; and theexogenous gene is selected from an NDV F gene, an AIV HA gene, and anIBDV VP2 gene.
 2. The recombinant HVT according to claim 1, wherein thespacer region between an HVT005 region and an HVT006 region of an HVTgenome is located between 8,867 nt and 9,319 nt of the HVT genome, andhas a nucleotide sequence set forth in SEQ ID NO:
 1. 3. The recombinantHVT according to claim 1, wherein the NDV F gene is a complete openreading frame (ORF) of a VII type NDV F gene, and has a cleavage sitereplaced with a cleavage site of a live vaccine (La Sota strain) ofNewcastle disease in chicken; the nucleotide sequence of the NDV F geneis set forth in SEQ ID NO: 2; and an expression cassette of the NDV Fgene is ligated successively by an mCMV promoter, the NDV F gene and anSV40 poly A.
 4. The recombinant HVT according to claim 1, wherein theAIV HA gene is a complete ORF of an H9N2 subtype AIV HA gene; the H9N2subtype AIV HA gene has a nucleotide sequence set forth in SEQ ID NO: 3;and an expression cassette of the H9N2 subtype AIV HA gene is ligatedsuccessively by an mCMV promoter, the exogenous gene and an SV40 poly A.5. A method for preparing the recombinant HVT according to claim 1,comprising the following steps: S1) construction of a CRISPR-Cas9plasmid: S1-1) designing an sgRNA sequence for the spacer region betweenan HVT005 region and an HVT006 region, wherein the sgRNA sequence is setforth in SEQ ID NO: 7, TCATATACTGAATCGTAGGG SEQ ID NO: 7; and S1-2)synthesizing a plus-strand HVT005/006-sgRNA-F and a minus-strandHVT005/006-sgRNA-R according to the designed sgRNA sequence, andconducting annealing to form a dsDNA, and inserting the dsDNA into avector to obtain the CRISPR-Cas9 plasmid; wherein sequences of theplus-strand HVT005/006-sgRNA-F and the minus-strand HVT005/006-sgRNA-Rare set forth in SEQ ID NO: 8 and SEQ ID NO: 9, respectively: NameSequence HVT005/006-sgRNA-F caccgCATATACTGAATCGTAGGG SEQ ID NO: 8HVT005/006-SgRNA-R aaacCCCTACGATTCAGTATATGc SEQ ID NO: 9

S2) constructing a donor vector plasmid comprising an expressioncassette of the exogenous gene; S3) construction and purification of arecombinant HVT: co-transfecting a pX459-sgA plasmid, the CRISPR-Cas9plasmid and the donor vector plasmid into primary chicken embryofibroblasts (CEFs), inoculating with an HVT, followed by culturing untilthe primary CEFs are cytopathic; and conducting purification to obtain aprimary recombinant HVT; and S4) removing a green fluorescent protein(GFP) in the primary recombinant HVT obtained in step S3) to obtain therecombinant HVT.
 6. The method according to claim 5, wherein step S2)comprises specifically the following steps: S2-1) inserting theexogenous gene into a pcDNA3.1-SfiI vector to obtain apcDNA3.1-SfiI-exogenous gene vector; and S2-2) ligating an expressioncassette of the exogenous gene in the pcDNA3.1-SfiI-exogenous genevector into a pT-sgA-GFP vector through an SfiI restriction site toobtain a pT-sgA-GFP-exogenous gene vector.
 7. The method according toclaim 5, wherein step S4) comprises specifically the following steps:transfecting a pcDNA3.1-Cre plasmid in the primary CEFs, inoculatingwith the primary recombinant HVT obtained in step S3) until the primaryCEFs are cytopathic, followed by purification through virus spotting toobtain a recombinant virus rHVT-exogenous gene.
 8. A method forpreventing avian influenza and/or Newcastle disease and/or infectiousbursal disease, comprising administering a vaccine comprising therecombinant HVT according to claim
 1. 9. A vaccine for preventing avianinfluenza and/or Newcastle disease and/or infectious bursal disease,comprising the recombinant HVT according to claim
 1. 10. A method forinserting an exogenous gene in an HVT, comprising inserting theexogenous gene into an HVT genome using a CRISPR-Cas9 technology;wherein the exogenous gene is inserted into a spacer region between anHVT005 region and an HVT006 region of the HVT genome; an insertion siteis located between 8,867 nt and 9,319 nt in the spacer region between anHVT005 region and an HVT006 region of the HVT genome, and has anucleotide sequence set forth in SEQ ID NO: 1; and more preferably, theCRISPR-Cas9 technology is achieved by construction of a CRISPR-Cas9plasmid: S1-1) designing an sgRNA sequence for the spacer region betweenan HVT005 region and an HVT006 region, wherein the sgRNA sequence is setforth in SEQ ID NO: 7, TCATATACTGAATCGTAGGG SEQ ID NO: 7; and S1-2)synthesizing a plus-strand HVT005/006-sgRNA-F and a minus-strandHVT005/006-sgRNA-R according to the designed sgRNA sequence, andconducting annealing to form a dsDNA, and inserting the dsDNA into avector to obtain the CRISPR-Cas9 plasmid; wherein sequences of theplus-strand HVT005/006-sgRNA-F and the minus-strand HVT005/006-sgRNA-Rare set forth in SEQ ID NO: 8 and SEQ ID NO: 9, respectively: NameSequence HVT005/006-sgRNA-F caccgCATATACTGAATCGTAGGG SEQ ID NO: 8HVT005/006-sgRNA-R aaacCCCTACGATTCAGTATATGc SEQ ID NO: 9


11. The method according to claim 5, wherein the spacer region betweenan HVT005 region and an HVT006 region of an HVT genome is locatedbetween 8,867 nt and 9,319 nt of the HVT genome, and has a nucleotidesequence set forth in SEQ ID NO:
 1. 12. The method according to claim 5,wherein the NDV F gene is a complete open reading frame (ORF) of a VIItype NDV F gene, and has a cleavage site replaced with a cleavage siteof a live vaccine (La Sota strain) of Newcastle disease in chicken; thenucleotide sequence of the NDV F gene is set forth in SEQ ID NO: 2; andan expression cassette of the NDV F gene is ligated successively by anmCMV promoter, the NDV F gene and an SV40 poly A.
 13. The methodaccording to claim 5, wherein the AIV HA gene is a complete ORF of anH9N2 subtype AIV HA gene; the nucleotide sequence of the H9N2 subtypeAIV HA gene is set forth in SEQ ID NO: 3; and an expression cassette ofthe H9N2 subtype AIV HA gene is ligated successively by an mCMVpromoter, the exogenous gene and an SV40 poly A.
 14. The methodaccording to claim 8, wherein the spacer region between an HVT005 regionand an HVT006 region of an HVT genome is located between 8,867 nt and9,319 nt of the HVT genome, and the nucleotide sequence of the insertionsite set forth in SEQ ID NO:
 1. 15. The method according to claim 8,wherein the NDV F gene is a complete open reading frame (ORF) of a VIItype NDV F gene, and has a cleavage site replaced with a cleavage siteof a live vaccine (La Sota strain) of Newcastle disease in chicken; thenucleotide sequence of the NDV F gene is set forth in SEQ ID NO: 2; andan expression cassette of the NDV F gene is ligated successively by anmCMV promoter, the NDV F gene and an SV40 poly A.
 16. The methodaccording to claim 8, wherein the AIV HA gene is a complete ORF of anH9N2 subtype AIV HA gene; the nucleotide sequence of the H9N2 subtypeAIV HA gene is set forth in SEQ ID NO: 3; and an expression cassette ofthe H9N2 subtype AIV HA gene is ligated successively by an mCMVpromoter, the exogenous gene and an SV40 poly A.
 17. The methodaccording to claim 9, wherein the spacer region between an HVT005 regionand an HVT006 region of an HVT genome is located between 8,867 nt and9,319 nt of the HVT genome, and has a nucleotide sequence set forth inSEQ ID NO:
 1. 18. The method according to claim 9, wherein the NDV Fgene is a complete open reading frame (ORF) of a VII type NDV F gene,and has a cleavage site replaced with a cleavage site of a live vaccine(La Sota strain) of Newcastle disease in chicken; the nucleotidesequence of the NDV F gene is set forth in SEQ ID NO: 2; and anexpression cassette of the NDV F gene is ligated successively by an mCMVpromoter, the NDV F gene and an SV40 poly A.
 19. The method according toclaim 9, wherein the AIV HA gene is a complete ORF of an H9N2 subtypeAIV HA gene; the nucleotide sequence of the H9N2 subtype AIV HA gene isset forth in SEQ ID NO: 3; and an expression cassette of the H9N2subtype AIV HA gene is ligated successively by an mCMV promoter, theexogenous gene and an SV40 poly A.